he concept of HSC-based gene therapy for HIV disease emerged in the epidemic’s first decade, when effective antiretroviral regimens were nonexistent. First, it was demonstrated that CD4+ T-cell depletion, the hallmark of HIV disease, was caused not simply by destruction of late-stage CD4+ T effector cells but also by the host’s inability to maintain progenitor cells, including HSCs, intrathymic T progenitor cells and central memory T cells in the periphery. Then, that HIV can persist within multiple lineages of long-lived cells, including T cells and cells of the myeloid lineage, some of which appear to be progenitor cells. However, early attempts to engineer HIV resistance into hematopoietic progenitor cells encountered insurmountable hurdles.

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ore recently, three important developments have prompted a reevaluation of HSC-based therapy for HIV:-a critical target, the cell-surface receptor CCR5, was identified;-a 32-base-pair deletion in the CCR5 gene, CCR5 delta 32, leading to a truncated gene product, was identified. Patients who are heterozygous for CCR5 delta 32 have delayed disease progression after they acquire HIV, whereas homozygotes rarely acquire HIV;-and an HIV-infected individual was reported to be virus free in the absence of ART after receiving a transplant of CCR5-defective allogeneic HSC.This set of observations inspired several groups to pursue CCR5-targeted gene therapy. One innovative approach relied on engineered zinc-finger nucleases specific for the CCR5 gene15. Such ‘molecular scissors’ can be delivered to cells ex vivo using methods such as integrase-defective lentiviral vectors, adenoviral vectors and plasmid DNA nucleofection. After specific binding of a pair of zinc-finger nucleases to the CCR5 gene, a double-stranded DNA break is introduced and then repaired by pathways that include error-prone non homologous end-joining. This can create a permanent gene disruption that is passed to daughter cells in the absence of persistent transgene expression. The result is the functional disruption of the CCR5 gene.A clinical trial is currently ongoing in humans using this approach in CD4+ T cells (see also on this website, the IHV Tropea meeting report). Although of great interest, this strategy does not disrupt CCR5 in HSCs and thus would not enable the long-term generation of both T and myeloid-lineage cells resistant to HIV infection.In a recent Nature Biotechnology paper (1), Holt et al. describe an experiment where human hematopoietic/progenitor stem cells (HSPCs) were isolated and treated with zinc-finger nucleases (ZFN) to dysrupt the CCR5 gene. Mice that were successfully engrafted with CCR5-disrupted HSPCs tolerated infection with HIV, whereas those engrafted with unmodified HSPCs exhibited loss of CD4+ T cells and high-level viremia (figure).

HSPCs is a population of cells enriched in HSCs. A mean of 17% of the cells were successfully modified, 5–7% of which were estimated to be homozygous CCR5–. Modified or unmodified CD34+ cells were then transplanted into 'NSG' mice, a model known to support multilineage human hematopoiesis (NOD/SCID/IL2rgammanull).As expected, mice engrafted with unmodified stem cells and subsequently challenged with CCR5-tropic HIV showed high levels of viremia and loss of peripheral and tissue human T cells. However, in animals repopulated with CCR5-disrupted HSPCs, the virus levels were lower and CD4+ T cells were not depleted, either in the peripheral blood or in the hematolymphoid tissues.

Transient ZFN treatment of human CD34+ HSPCs was found efficient to disrupt CCR5 while yielding cells that remain competent to engraft and support hematopoiesis. In the presence of CCR5-tropic HIV-1, CCR5−/− progeny rapidly replaced cells depleted by the virus, leading to a polyclonal population that ultimately preserved human immune cells in multiple tissues.The frequency of cells containing evidence of CCR5 disruption increased to >80% in the peripheral blood and to >40% in multiple tissues by week 12 of infection.

This indicates that the modification of only a minority of human CD34+ HSPCs may provide the same strong anti-viral benefit as was conferred by a complete CCR5 delta 32 stem cell transplantation in a patient.

The observation of almost complete replacement of human T cells in the intestines of the infected mice with CCR5– cells is consistent with this tissue harboring the majority of the body’s CD4+CCR5+ effector memory cells. A strong selection for CCR5– cells was also observed in the thymus, suggesting that CCR5– cells would be selected at both a precursor stage in the thymus and at an effector stage in the mucosa.Ultimately, the presence of HIV-resistant CCR5– cells in mucosal tissues should both protect individual cells from infection and help to break the cycle of immune hyperactivation that may underlie much of the pathology of AIDS.

This paper was introduced by an outstanding Editorial from Steve Deeks (2) who qualified these results as 'convincing'. He also detailed the technical issues raised by these findings and the next steps to be taken.

Technical issues:

Do genetically modified HSCs confer benefit to a mouse that is already infected?

Does a CCR5-disrupted hematopoietic compartment confer protection against infection by X4 variants?

Is the CCR5-disrupted immune system normal?

Next steps to be addressed:

Can we allow HIV to replicate at high levels in the absence of ART so that CCR5-deficient cells can be selected, knowing that HIV replication is harmful (inflammation...)?

Will resistant viruses (like X4) be selected?

Will this therapy be beneficial to most advanced patients who already have dual tropic strains?

Will ablative therapy be needed to allow stem celle engraftment?

Will this be sufficient to conteract CD4 depletion as this one is not uniquely the result of direct viral killing?

Finally, Deeks points out that such a new approach should be affordable to all, and that a reprioritization is needed at a governmental level for a global program motivated by a common desire for a world free of HIV.